Biochemical characterization of nitrate transport at the plasma membrane of plant cells using isolated vesicles
Ruiz-Cristin, Jose-Luis
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https://hdl.handle.net/2142/19009
Description
Title
Biochemical characterization of nitrate transport at the plasma membrane of plant cells using isolated vesicles
Author(s)
Ruiz-Cristin, Jose-Luis
Issue Date
1990
Doctoral Committee Chair(s)
Briskin, Donald P.
Department of Study
Crop Sciences
Discipline
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture
Chemistry
Agronomy
Biochemistry
Language
eng
Abstract
A modified method for the production and purification of $\sp{36}$ClO$\sb3\sp{-}$ in high yield is described. This procedure, involving the elctrolytic oxidation of $\sp{36}$Cl in a cell with simple electrode design and purification of the electrolysis products ($\sp{36}$Cl, $\sp{36}$ClO$\sb3\sp{-}$ and $\sp{36}$ClO$\sb4\sp{-}$) by aqueous column chromatography in Sephadex G-10, allows for the recovery of about 80% of the initial radioactivity as $\sp{36}$ClO$\sb3\sp{-}$. This radiolabeled anion was used as a tracer for nitrate in membrane transport studies with isolated plasma membrane vesicles.
A nitrate/chlorate transport activity associated with the plasma membrane vesicles isolated from red beet storage tissue has been identified and characterized. This transport activity is inhibited by the protein modified reagent phenylglyoxal (PGO) and it is mediated by a uniport-type carrier driven by the membrane potential. Given the prevailing conditions of the electrochemical gradient across the membrane of plant cells, this activity seems to correspond with efflux from the intact plant cell. It is proposed that this transport activity is involved in the translocation of nitrate accumulated in the storage tissue to the growing shoot during the second season of this biennial plant.
Nitrate/chlorate transport in maize root plasma membrane vesicles is mediated by a symport-type carrier driven by the proton gradient across the membrane ($\Delta$pH). This activity seems to correspond to the cell uptake system most likely involved in nitrate acquisition. The protein modifier phenylglyoxal also inhibited this activity. Kinetic studies showed that protons, in addition to acting as the driving force for nitrate/chlorate uptake into the vesicles, changed the affinity of the carrier for the solute (Km) with no effect on Vmax, suggesting an ordered reaction mechanism for the carrier. This mechanism would involve initial H$\sp{+}$ binding to the carrier followed by the binding of nitrate/chlorate.
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